Macrocyclic antibiotics

ABSTRACT

The invention relates to cyclic antibiotics of formula ##STR1## wherein the prefix R represents that the substituents on the adjacent carbon atom are in the R configuration; the prefix S represents that the substituents on the adjacent carbon atom are in the S configuration; R 1  is hydrogen or hydroxy and R 2  is hydroxy or β-glucopyranosyloxy, and to the hydrates and salts thereof, especially pharmaceutically acceptable salts. 
     The compounds of formula I are prepared by fermentation using the strain So ce 12 (NCIB 12134) of the species Sorangium celulosum and are effective antiobiotics.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to novel macrocyclic compounds, inparticular to sorangicin A, to a fermentation process for thepreparation of said compounds using a novel microorganism of the speciesSorangium cellulosum (also known as Polyangium cellulosum) and to thenovel microorganism itself. The invention further relates topharmaceutical compositions which contain the novel compounds, to atherapeutic method comprising the use of said compounds as antibiotics,and to the use thereof for the preparation of pharmaceuticalcompositions.

SUMMARY OF THE INVENTION

Specifically, the present invention relates to macrocyclic compounds offormula ##STR2## wherein the prefix R represents that the substituentson the adjacent carbon atom are in the R configuration; the prefix Srepresents that the substituents on the adjacent carbon atom are in theS configuration; R₁ is hydrogen or hydroxy and R₂ is hydroxy orβ-glucopyranosyloxy, and to solvates and salts thereof, in particular topharmaceutically acceptable acid addition salts thereof.

The configuration of the substituents at the chiral C-atoms has beenascertained by means of X-ray structural analysis of the crystallinecompound of formula I, sorangicin A (R₁ =OH, R₂ =OH), and determined bythe sequence rules of Cahn, Ingold and Prelog. If R₁ is hydroxy, theC-22 atom has the S-configuration.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates in particular to the main component of formula Iobtainable by fermentation and designated as sorangicin A (R₁ =OH, R₂=OH, C-22:S), and to the secondary components comprising sorangicin B(R₁ =H, R₂ =OH), sorangioside A (R₁ =OH, R₂ =β-glucopyranosyloxy,C-22:S), and sorangioside B (R₁ =H, R₂ =β-glucopyranosyloxy).

A further preferred object of the invention is the depositedmicroorganism of the species Sorangium cellulosum, Polyangium cellulosumwhich is described hereinafter and is employed for the preparation ofthe main component, sorangicin A, by fermentation, as well as the of thesecondary components cited above. The taxa "Sorangium cellulosum" asused herein is synonymous with "Polyangium cellulosum", which terms areused interchangeably in the scientific community.

The compounds of formula I may be in the form of solvates. For example,sorangicin A can be isolated in the form of the crystalline ethylacetate solvate.

Salts are preferably the pharmaceutically acceptable or non-toxic saltsof compounds of formula I. Such salts are in particular suitable alkalimetal salts such as sodium or potassium salts, or alkaline earth metalsalts, e.g. magnesium or calcium salts, and also zinc salts or ammoniumsalts, including those salts that are formed with organic amines such asunsubstituted or hydroxy-substituted mono-, di- or trialkylamines, e.g.diethylamine, bis(2-hydroxyethyl)amine, triethylamine,N,N-dimethyl-N-(2-hydroxyethyl)amine, tris(2-hydroxyethyl)amine orN-methyl-D-glucamine. Pharmaceutically unacceptable salts, e.g.sparingly soluble and/or readily crystallising salts can be used forisolation and purification.

The compounds of formula I, especially the main component, sorangicin A,which is obtainable by fermentation, as well as pharmaceuticallyacceptable salts thereof, are effective antibiotics for use in human andveterinary medicine. Their activity is directed against coccae, bacteriaand viruses. Controllable viruses are in particular those requiringreverse transcriptase for replication (retroviruses).

For example, MIC (minimum initiatory concentration) values have beenfound in vitro in the agar dilution test (H. M. Ericsson and S. C.Sherris, Acta Path. Microb. Scand. Section B, Suppl. No. 217, 1-90,1971) which are in the range from 0.01 to 2 μg/ml for aerobic,gram-positive and gram-negative coccae, e.g. Staphylococcus aureus,Neisseria gonorrhoeae, N. meningitidis, Streptococcus spp. and from c.0.01 to 16 μg/ml for Haemophilus influenzae, Pseudomonas aeruginosae,enterobacteria such as Escherichia coli, Klebsiella pneumoniae, Proteusspp., Enterobacter cloacae, Serratia marcescens or anaerobes, e.g.Bacteroides fragilis or Clostridium perfingens. MIC values in the rangefrom 0.5 to 8 μg/ml have been found for mycobacteria, e.g. M.tuberculosis and atypical mycobacteria.

In the in vitro test on human polymorphonuclear leucocytes and piStaphylococcus aureus Strain Wood 46, sorangicin A is effective againstbacteria that occur intracellularly, e.g. staphylococcae. The substancehas a bactericidal activity within leucocytes.

Proof of the antiviral activity of the compounds of formula I againstretroviruses is shown by in vitro and in vivo tests, but especially byan in vitro test system in which the inhibition of the reversetranscriptase enzyme is determined quantitatively by said compounds.

On poly A-oligo (dT), radioactively labelled TPP is hybridised withreverse transcriptase. The latent radioactivity is measured and servesas reference for the activity of the reverse transcriptase.

The ID₅₀ of the compounds of formula I is determined by using them inthe test system in different concentrations.

Sorangicin A is active against reverse transcriptase of Moloney Murineleucaemia virus; the ID₅₀ dose that inhibits the RT activity by 50%;method of Wu et al., Proc. Natl. Acad. Sci., 69, 3820 (1972)] for thereverse transcriptase of MLV is c. 7 μg/ml.

Compounds of formula I are prepared by culturing the strain So ce 12(NCIB 12134) of the species Sorangium cellulosum, or a mutant derivedfrom said strain that produces compounds of formula I, in a culturecontaining a source of carbon and nitrogen and essential inorganicsalts, in the temperature range from about 15° to 40° C. and at a pH inthe range from about 4.0 to 8.0, under aerobic conditions and, ifdesired, isolating the resultant compounds of formula I and/orconverting a resultant compound into a salt and/or a resultant salt intothe free compound or into another salt.

The microorganism So ce 12 of the species Sorangium cellulosum wasobtained from a soil sample from the region of Xcaret, YucatanPeninsula, Mexico. It can be classified as a strain of themyxobacterium, Sorangium cellulosum. The public has access to thedeposit.

The strain So ce 12 was deposited on lst August 1985 with The NationalCollections of Industrial and Marine Bacteria Ltd. in Aberdeen,Scotland, UK, under the number NCIB 12134, in accordance with the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure. A viabilitycertificate was issued by this depository authority on 12th August 1985.

Isolation and description of So ce 12

The soil sample was placed on filter paper over Stan 21 agar (0.1% of K₂HPO₄, 0.1% of KNO₃, 0.1% of MgSO₄.7H₂ O, 0.01% of MnSO₄. 7H₂ O, 0.1% ofCaCl₂.2H₂ O. 0.02% of FeCl₃, standard trace element solution (consistinge.g. of 20 mg/l each of Na₂ MoO₄.2H₂ O, Na₂ B₄ O₇.10 H₂ O, MnSO₄.H₂ Oand CuSO₄.H₂ O), 0.002% of yeast extract (Difco), 1% of agar, 25 mg/l ofcycloheximide (actidione), and incubated for 3 weeks at 30° C.

Swarm cell colonies with fruiting bodies were again inoculated on filterpaper over Stan 21 agar. Amoebae were removed by aeration with a 5%aqueous solution of ammonia. The fruiting bodies in EBS solution (0.5%of peptone from casein, tryptic, ex Merck Darmstadt, Germany, 0.5% ofproteosepeptone, Difco, 0.1% of peptone from meat extract, Merck, 0.1%of yeast extract, Difco, sterilised in an autoclave) were then keptovernight at 4° C. with AB-1 antibiotic solution (20 mg ofchloroamphenicol, 30 mg of streptomycin sulfate, 25 mg of tetracyclinehydrochloride, 20 mg of cephalotin, dissolved in 50 ml of water andsterilised by filtration) and subsequently streaked out on vy/2 agar(0.5% of baker's yeast, based on fresh weight, 0.1% of CaCl₂.2H₂ O, 1.5%of agar, pH 7.2). Developing, pure swarm cell colonies were furthercultivated on the same medium and examined on filter paper over Stan 21agar for identity with the starting isolate.

The vegetative cells are cylindrical rods with round ends, mostly c. 1μm wide and 3-6 μm long. Under a phase contrast microscope they appeardark. They move by gliding. On many nutrient media, the organism forms amultitude of fruiting bodies, e.g. on filter paper over mineral saltagar (Stan 21 agar). The fruiting bodies resemble rust-coloured pads andconsist of a substantially large number of sporangioles, spherical or,through mutual flattening, polyhedral structures with a solid wall 20 to30 μm in diameter.

The sporangioles contain myxospores, rod-shaped permanent cells ofsimilar shape and size as the vegetative cells, but readily refractive.

The strain So ce 12 can spontaneously form mutants (natural mutants orartificial mutants can be prepared which, like the natural strain,produce antiobiotic compounds of formula I.

Such mutants can be produced by chemical means, e.g. by treatment withcertain guanidine derivatives, e.g.N-methyl-N'-nitro-N-nitrosoguanidine, or with an alkali metal nitritesuch as sodium nitrite, or by physical means, e.g. with energy-richradiation such as ultraviolet, X-ray or radioactive radiation.

The culture eligible for culturing must contain a source of carbon andof nitrogen as well as essential inorganic salts. Examples of suitablecarbon sources are: assimilable carbohydrates, e.g. D-glucose, D-xylose,L-arabinose, D-fructose, maltose, maltotriose, and starch. Suitablenitrogen sources are: amino acids, peptides and proteins as well astheir degradation products such as peptone or tryptone, and also meatextracts, cereal flour, e.g. corn or wheat, beans, especially soybeans,seeds, e.g. of cotton plants, distillation residues from alcoholproduction, yeast extracts and the like, and ammonium salts andnitrates. As essential inorganic salts the nutrient solution may containe.g. chlorides, carbonates, sulfates, phosphates of alkali metals oralkaline earth metals, e.g. sodium, potassium, magnesium, calcium, iron,zinc, manganese, molybdenum and copper. Culturing is preferably carriedout in liquid culture media, most preferably in aqueous culture media.

A particularly suitable culture medium is MD1 medium (peptone fromcasein, tryptic (Merck), 0.3%; CaCl₂.2H₂ O, 0.05%; MgSO₄.7H₂ O, 0.2%;which is enriched with a carbohydrate source, e.g. glucose, maltose,maltotriose, starch, cellulose, each 0.1%.

A further useful liquid culture medium contains peptone from casein,0.1%; CaCl₂.2H₂ O, 0.05%; MgSO₄.7H₂ O, 0.2%; corn steep powder or zein,0.4%. The strain can also be cultivated in a defined medium consistingof MgSO₄.7H₂ O, 0.15%; FeCl₃.6H₂ O, 8 mg/l; KNO₃, 0.2%; K₂ HPO₄, 0.025%;glucose.H₂ O, 0.5%; CaCl₂.2H₂ O, 0.15%. Addition of 0.01-0.05% ofpeptone results in homogeneous growth, an improved cell yield, and inincreased antibiotic production.

Culturing can be effected batchwise, e.g. by single or repeated additionof nutrient solution, or continuously by continuous addition of nutrientsolution.

It is preferred to culture in several stages by preparing a preculture(inoculum), e.g. in one of the cited culture media, with whichpreculture the actual main culture is subsequently inoculated afterfermentation for one or two days, preferably in a dilution ratio of1:10.

This preculture is obtained for example from a series of precultures.The first culture of this series is obtained by growing the strain inquestion for 14 days on a solid or liquid nutrient medium, e.g.agar+MDl+0.1 % of starch. A nutrient solution is then inoculated withthe culture and incubated for several days, e.g. 4-5 days. This nutrientsolution can, if desired, be used for inoculating a further nutrientsolution, e.g. MD1+0.1% of starch, or further nutrient solutions atintervals of 4-5 days, e.g. in a dilution ratio of about 3% (v/v), andthe batch is incubated under the stated conditions.

The course of the fermentation can be monitored analytically during thefermentation, e.g. by measuring the pH value of the culture, whichduring the fermentation falls from about 7.2 to about 6.0-6.5 and thenrises to about 7.5-8.0, or the optical density, which is a referencestandard for determining the growth of the particular strain, as well asgravimetrically on the basis of the dry weight of the resultant biomass,by thin-layer chromatography or by determining the antibiotic activityof the components present in the culture filtrate.

The isolation of the compounds of formula I, especially of the maincomponent, sorangicin A, from the fermentation broth is effected bymethods known per se, having regard to the chemical, physical andbiological properties of the substances. Determination of theconcentration of antibiotics in the individual isolation steps, as wellas in the culture medium, can be made by thin-layer chromatography, e.g.on silica gel (elution with e.g. methylene chloride/methanol), and/or bythe activity against different microorganisms, e.g. Staphylococcusaureus, and/or by the inhibition of reverse transcriptase.

Two methods are suitable for isolating the compounds of formula I fromthe crude fermentation broth:

a) Stirring the fermentation broth with macroporous non-ionic adsorberresins, e.g. synthetic resins of aromatic structure, for example resinsbased on polystyrene, e.g. styrene/divinylbenzene copolymers. Suchresins can be characterised by different customary statistical data suchas pore volume, specific surface area, average pore diameter, mostfrequent pore diameter, pore size distribution, bead size distributionand the like.

Suitable adsorber resins have a pore volume of about 0.5 to 4.5 ml/g, aspecific surface area of about 100-1000 m₂ /g, and an average porediameter of about 4 to 130 nm, and are commercially available under theregistered trademarks AMBERLITE XAD-1, XAD-2, XAD-4, XAD-1180 and ER-180ex Rohm & Haas, DIAION HP-10, HP-20, HP-21, HP-30, EP-40, HP-50 exMitsubishi, DUOLITE S-861, S-862, S-863 and ES 866 ex Dia-Prosim, IMACSyn 46 and Syn 72 ex Akzo Chemie, KASTEL S-111, S-112, S-114 exMontedison, LEWATIT OC.1031 ex Bayer and RELITE ADS ex Resindion.

After separating the fermentation broth from the adsorber resin, e.g. bysieving, the resin is washed with water and subsequently eluted withincreasing amounts of an organic solvent which is inert to the adsorberresin, e.g. a mixture of water/methanol, with the bulk of the compoundsof formula I being eluted with 80-100% methanol. The eluates areconcentrated in vacuo and, as described for variant b), separated byHPLC.

(b) The fermentation broth is separated from the biomass in conventionalmanner, e.g. by filtration or centrifugation, and the filtrate isextracted with a water-immiscible, or substantially water-immiscible,solvent, e.g. methylene chloride, chloroform or, preferably, ethylacetate. The organic phase is evaporated under vacuum, affording a crudeextract. This crude extract is partitioned in a two-phase systemconsisting of two immiscible organic solvents, e.g. methanol/heptane,and the fermentation products, in particular the antibiotics of formulaI, become enriched in the polar phase. After removal of the polarsolvent by evaporation, the residue is dissolved in dilute ammoniasolution and washed with a water-immiscible solvent, e.g. diethyl ether.The volatile ammonia is removed under vacuum and the aqueous phase,after acidification with an organic acid, e.g. formic acid or aceticacid, is extracted with one of the water-immiscible, or substantiallywater-immiscible, solvents mentioned above, e.g. methylene chloride. Theresultant antibiotics are subsequently purified by reversed phasechromatography, affording several fractions which may containsorangioside A and B in admixture or in pure form, and sorangicin A andB in admixture or in pure form. The individual components sorangioside Aand B and sorangicin B are obtained by separation of the respectivefractions by HPLC, whereas the main component, sorangicin A, can beobtained in crystalline form from the appropriate fraction.

The main component, sorangicin A, can also be obtained by other variantsof these known separation methods, e.g. by using other solvents ormixtures of solvents, or by using other chromatographic methods.

Salts of compounds of formula I can be prepared in a manner known perse. Thus it is possible to form salts of compounds of formula I e.g. bytreatment with metal compounds such as alkali metal salts of suitableorganic carboxylic acids, e.g. the sodium salt of 2-ethylhexanoic acid,or with an inorganic alkali metal salt or alkaline earth metal salt,e.g. sodium bicarbonate, or with ammonia or a suitable organic amine,using the salt-forming compound preferably in stoichiometric amount oronly a small excess thereof.

Salts can be converted in conventional manner into the free compounds,metal and ammonium salts e.g. by treatment with a suitable acid.

Mixtures of stereoisomers, especially of diastereoisomers, can beseparated into the individual isomers in a manner known per se, e.g. byfractional crystallisation, chromatography and the like.

Racemates can be resolved in a manner known per se, e.g. afterconverting the optical antipodes into diastereoisomers, for example byreaction with an optically active acid or base or with specificmicroorganisms.

The invention also relates to those embodiments of the process in whichan intermediate obtainable in any stage of the process is used asstarting material and the remaining steps are carried out therewith, orthe process is interrupted at any stage or a compound obtainable by theprocess of the invention is prepared under the conditions of the processand further processed in situ.

The pharmacologically acceptable compounds of the present invention canbe used e.g. for the preparation of pharmaceutical compositions whichcontain an effective amount of a compound of formula I, preferably inadmixture with a significant amount of inorganic or organic, solid orliquid pharmaceutically acceptable excipients. The invention alsorelates to such pharmaceutical compositions and to the preparation anduse thereof.

The pharmaceutical compositions of this invention are suitable forparenteral, e.g. intravenous or intramuscular, administration, and, ascircumstances may require also for oral administration or topicalapplication.

The compounds of formula I are used, for example, in the form ofinjectable compositions, e.g. for intravenous administration, or ofinfusion solutions. Such solutions are preferably isotonic aqueoussolutions or suspensions which can be prepared prior to use, e.g. fromlyophilised preparations which contain the active ingredient alone ortogether with a carrier, e.g. mannitol. Pharmaceutical compositions fororal administration may be sterilised and can contain adjuvants, e.g.preservatives, stabilisers, wetting agents and/or emulsifiers,solubilisers, salts for regulating the osmotic pressure, resorptionpromoters and/or buffers. The pharmaceutical compositions of thisinvention which, if desired, may contain further useful pharmacologicalsubstances, e.g. other active ingredients, contain about 0.1 to 100%,preferably about 1 to 100%, of active ingredient.

The pharmaceutical compositions are prepared in a manner known per se,e.g. by conventional dissolving or lyophilising methods described intextbooks of pharmacology.

Utility

Compounds of formula I, and the solvates or pharmaceutically acceptablesalts thereof, can be used as antibiotics in the form of pharmaceuticalcompositions for the therapeutic treatment of the human or animal body,for example as antibacterial antibiotics for the treatment of infectionscaused by gram-positive or gram-negative bacteria. e.g. by Neisseriagonorrhoeae or meningitidis, Staphylococcus aureus or epidermis,streptococcae, including enterococcae, Haemophilus influenzae.Pseudomonas aeruginosae, or anaerobic pathogens such as Bacteroidesfragilis or Clostridia spp., infections that are caused by bacteria thatsurvive in cells, e.g. leucocytes, for example staphylococcae orlisteriae, tuberculosis or infections that are caused by atypicalmycobacteria such as Mycobacterium intracellulare avium.

The compounds of this invention can also be used as antiviralantibiotics, in particular for the treatment of infections caused byviruses with reverse transcriptase activity, e.g. retroviruses(including HTLV III that causes AIDS), or hepatitis viruses and thelike. In addition to infections with HTLV III (also named LAV I or HIV),other infections which can be treated with the compounds of formula Iare those with HTLV I, HTLV II and HTLV IV (also named LAV II).

Depending on the nature, severity and duration of the infection, thecondition of the patient and the mode of administration, daily doses ofabout 0.3 g to 10.0 g will be administered subcutaneously,intramuscularly, intravenously, orally or by inhalation, for thetreatment of persons or warm-blooded animals having a body weight of 70kg.

Further utilities exist in the field of plant protection, e.g. forcontrolling bacterial diseases of greenhouse plants, and for the care ofinfected trees and the like. Aerosols are suitable for such utilities.

The following Examples illustrate the experimental reduction to practiceand general operability of the present invention.

EXAMPLE 1 (a) Preparation of the inoculum of the preculture Bacteria ofthe strain So ce 12 in 100 ml of liquid MD1 medium taken from an approx.14-day-old agar plate containing the medium MD1 (0.3% of peptone fromcasein, tryptic (ex Merck, Germany), 0.05% of CaCl₂.7H₂ O, 0.2% ofMgSO₄.7H₂ O) and 0.1% of starch are inoculated with 0.1% of starch andthe culture is incubated on a gyratory table at 30° C. and 160 rpm(revolutions per minute). After incubation for 5 days, the culture isadded to a fresh culture medium MD1 in a dilution ratio of 3% (v/v).Fresh nutrient medium MD1 can be inoculated as often as required in adilution ratio of 3% (v/v), in each case after incubating the preculturefor 4 days. The last preculture of this series is used in a dilutionratio of 10% (v/v) as inoculum for 4 l of a further preculture with MD1nutrient solution. This preculture is incubated at 30° C., stirredbriefly and aerated under a weak flow of air. After incubation for 2days, a 70% fermentsr is inoculated with this inoculum for thepreparation of the preculture proper. (b) Preparation of the preculture

70 l fermenter (available from Giovanola, Monthey VS, Switzerland),equipped with paddle stirrer system. MD1 medium (1.0 m) with 0.1%starch. Temperature 30° C. Stirring speed 320 rpm. Air supply: 0.3 Nm²/h. The percentage of oxygen partial pressure falls in the course of thefermentation and is 10-20% after 2 days. OD₆₂₃ : c. 2. A 700 l fermenteris inoculated with this preculture under sterile conditions.

(c) Main culture

700 l fermenter equipped with paddle stirrer system available from thesame firm as in (b). Medium: 0.15% of magnesium sulfate heptahydrate, 8mg/l of iron(III) chloride hexahydrate, 0.05% of peptone, 0.2% of KNO₃,0.025% of K₂ HPO₄, 0.5% of glucose, 0.15% of calcium chloride dihydrate;pH 7.3; initial stirring speed: 150 rpm, after 23 hours: 100 rpm.Percentage of oxygen partial pressure at the conclusion of thefermentation: 10%. The fermentation is complete after 40 hours. Uponconclusion of the fermentation, the supernatant has an inhibiting zonediameter against Staphylococcus aureus of c. 14-15 mm. The biomass isseparated from the fermentation broth by centrifugation. The antibioticcomponents are present in the culture filtrate. All process steps arecarried out under sterile conditions.

(d) Working up of the culture filtrate

The culture filtrate is extracted with ethyl acetate. The solvent isthen evaporated, affording a crude extract. This extract is partitionedin a two-phase system consisting of heptane/methanol, and the compoundsof formula I become enriched in the methanolic phase. The methanol isremoved by evaporation and the residue is dissolved in 2% aqueousammonia solution. The ammoniacal solution is washed repeatedly withether and freed from ammonia under vacuum. The aqueous phase isacidified with formic acid and extracted with methylene chloride. Theextract is concentrated by evaporation and separation of the residue iseffected by reversed phase chromatography. Column: Labochrom PGC column674×37 mm (available from Labomatic), packed with LiChroprep RP-18(25-40 pm), ex Merck; eluant: methanol/buffer 68:33, buffer=0.5% formicacid in water, adjusted to pH 7 with triethylamine. Rate of flow: 30ml/min; detection: UV absorption at 313 nm. The following fractions areobtained:

fraction 1: peak at t_(R) =31 min: sorangiosid A+B

fraction 2: peak at t_(R) =40 min: sorangiosid B

fraction 3: peak at t_(R) =50 min: sorangicin A

fraction 4: peak at t_(R) =69 min: sorangicin B

After removal of the organic solvent, the components present in theindividual fractions are extracted from the aqueous buffered phase withdichloromethane or ethyl acetate.

Fraction 1: The mixture of sorangioside A and B is separated by HPLC:column 250×16 mm (Knauer), packed with LiChrosorb Si 100 (10 μ), exMerck; detection by UV absorption at 313 nm, eluant:dichloromethane/heptane/isopropanol/conc. buffer (52:4:7:1), conc.buffer consisting of a 1:2 mixture of methanol/formic acid neutralisedwith triethylamine; rate of flow: 18 ml/min. Sorangioside B is obtainedat a retention time t_(R) =9.4 min and sorangioside A at a retentiontime t_(R) =13.8 min.

Sorongioside A:

HPLC: column 250×16 mm (Knauer), packed with Nucleosil 7 C₆ H₅ (exMacherey-Nagel), eluant: methanol/water (65/35)+0.5% formic acid; rateof flow: 10 ml/min; detection: UV absorption at 313 nm: t_(R) =26.4 min.

Thin-layer chromatography (silica gel Si 60 F 254 (Merck), eluant:methylene chloride/methanol (8:2): R_(f) =0.44.

[α]_(D) ²² =+42.0 (c=0.9 in methanol)

UV (in methanol): λ_(max) (1 g ε)=301 (4.34) ¹³ C-NMR, see Table.

Fraction 2: The fraction containing sorangioside B is purified by HPLC:column 250×16 mm (Knauer), packed with Nucleosil 7 C₆ H₅ (ex Macherey-Nagel); detection: UV absorption at 131 nm; eluant: methanol/water(70:30)+0.5% formic acid; rate of flow: 14 ml/min; t_(R) =10.2 min.

Sorongioside B:

Thin-layer chromatography (silica gel Si 60 F 254 (Merck), eluant:methylene chloride/methanol (8:2): R_(f) =0.62.

[α]_(D) ²² =+5.1 (c=1.8 in methanol)

UV (in methanol): λ_(max) (1g ε)=301 (4.41) ¹³ C-NMR, see Table.

Fraction 3: Crystallised from ethyl acetate.

Sorangicin A:

m.p.: 105°-107° C.

[α]_(D) ²² =+60.9 (c=0.7 in methanol)

UV (MeOH): λ_(max) (1 gε)=301 (4.33)

¹³ C-NMR see Table

FAB-MS [neg. ions, xenon at 9 keV (Iontect), acceleration 8 kV,post-acceleration 11 KV]: m/e=805 (M-H)⁻ 897 (M-H+glycerol)⁻

EI-MS (70 eV, 245°): m/e %=806 (M⁺, 0.3), 789 (0.6), 788 (1.5), 770(0.8), 373 (2), 303 (2.5), 301 (3.5), 249 (44), 231 (5), 197 (15), 149(20), 135 (21), 133 (22), 121 (35), 109 (40), 107 (43), 105 (100)

The compound is present in the form of the ethyl acetate. It can becrystallized also from acetone.

Analysis

C₄₇ H₆₆ O₁₁ ×C₄ H₈ O theory C 6843 H 8.33, (0 23.23, found C 68.11 H8.35 (0 23.61.

Fraction 4: Purification by HPLC: column 250×16 mm (Knauer), packed withNucleosil 7 C₆ H₅ (ex Macherey- Nagel); detection: UV absorption at 313nm; eluant: methanol/water (75:25)+0.5% formic acid; rate of flow: 14ml/min; t_(R) =11.7 min.

Sorangicin B:

[α]_(D) ²² =+49,1 (c=1.6 in methanol)

UV (methanol): λ_(max) (1 gε)=301 (4,41)

¹³ C-NMR see Table

FAB-MS [neg. ions, xenon at 9 keV (Iontect), acceleration -8 kV,post-acceleration 11 kV]: m/z=789 (M--H)⁻, 881 (M--H+glycerol)⁻

EI-MS (70 eV, 270°): m/z (%)=790 (M⁺,1), 773 (3), 772 (5), 755 (2), 754(3), 643 (1), 250 (20), 249 (100), 197 (22), 191 (11), 181 (12), 169(10), 163 (11), 161 (11), 159 (11), 149 (22), 133 (22), 123 (23), 121(29), 107 (31), 105 (61) High

High resolution

C₄₇ H₆₆ O₁₀ theory 790,4656 found 790,4618.

                  TABLE                                                           ______________________________________                                        .sup.13 C-NMR data of the sorangicins and sorangiosides in CD.sub.3 OD        No.      Sorangioside.sup.d                                                                            Sorangicin                                           C-Atom   A        B          A      B                                         ______________________________________                                         1       178.01s  177.69s    177.85s                                                                              177.72s                                    2       35.38t   35.14t     35.28t 35.20t                                     3       26.27t   26.27t     26.29t 26.54t                                     4       28.20t   28.18t     28.20t 28.21t                                     5       38.52t   38.34t     38.51t 38.24t                                     6       32.90d   32.98d     32.96d 33.02d                                     7       a        s          a      a                                          8       131.26s  131.68s    131.24s                                                                              131.48s                                    9       74.44d   74.72d     74.42d 74.45d                                    10       66.95d   67.03d     66.90d 66.91d                                    11       123.91d  123.70d    123.79d                                                                              123.68d                                   12       136.94d  136.89d    136.85d                                                                              136.86d                                   13       75.37d   74.72d     75.33d 75.07d                                    14       b        b          b      b                                         15       a        a          a      a                                         16       a        a          a      a                                         17       b        b          b      b                                         18       b        b          b      b                                         19       136.41d  134.42d    134.36d                                                                              137.37d                                   20       127.23d  132.41d    130.16d                                                                              132.88d                                   21       83.21d   80.45d     74.36d c                                         22       76.04d   43.05t     77.77d 45.08t                                    23       74.05d   71.10d     74.87d c                                         24       31.40t   35.41t     30.86t 35.40t                                    25       70.98d   70.82d     71.07d 71.11d                                    26       38.03d   37.81d     38.51d 38.42d                                    27       74.44d   74.18d     75.05d 74.71d                                    28       b        b          b      b                                         29       a        a          a      a                                         30       a        a          a      a                                         31       81.15d   81.06d     81.17d 81.09d                                    32       42.11d   42.02d     42.15d 42.10d                                    33       81.06d   80.94d     81.03d 80.95d                                    34       39.80t   39.93t     39.85t 39.92t                                    35       77.53d   77.12d     77.59d 77.35d                                    36       82.03d   81.77d     82.26d 82.13d                                    37       134.91d  134.77d    134.88d                                                                              134.66d                                   38       127.76d  127.22d    127.84d                                                                              127.55d                                   39       137.57d  137.70d    137.57d                                                                              137.72d                                   40       127.00d  126.82d    126.98d                                                                              126.89d                                   41       139.14d  139.39d    139.05d                                                                              139.21d                                   42       119.72d  119.44d    119.69d                                                                              119.49d                                   43       167.67s  167.33s    167.68s                                                                              167.51s                                   44       21.73q   21.54q     21.67q 21.54q                                    45       14.30q   14.24q     14.30q 14.23q                                    46       10.62q   10.62q     10.89q 10.82q                                    47       15.36q   15.32q     15.36q 15.33q                                    ______________________________________                                         Footnotes:                                                                    a sorangioside A: 134.28d, 133.77d, 133.06d, 132.41d, 128.31d;                sorangioside B: 135.06d, 133.76d, 133.11d, 132.41d, 128.40d; sorangicine      A: 134.15d, 133.61d, 133.00d, 132.78d, 128.34d; sorangicine B: 134.27d,       133.53d, 133.03d, 132.70d, 128.40d;                                           b sorangioside A: 37.06t, 35.38t, 34.37t, 33.61t; sorangioside B: 37.01t,     35.41t, 33.85t, 33.64t; sorangicine A: 37.12t, 35.45t, 33.97t, 33.35t;        sorangicine B: 37.13t, 35.40t, 33.49t, 33.49t;                                c 72.13d, 71.72d;                                                             d glucosyl signals: C1' C2' C3' C4' C5' C6                               

EXAMPLE 2

Dry-filled ampoules or vials containing 0.5 g of sorangiosin A as activeingredient can be prepared as follows:

    ______________________________________                                        Composition: (for 1 ampoule or vial)                                          ______________________________________                                        active ingredient      0.5 g                                                  mannitol               0.05 g                                                 ______________________________________                                    

A sterile aqueous solution consisting of active ingredient and mannitolis filled under aseptic conditions into 5 ml ampoules or 5 ml vials,which are then sealed and tested.

What is claimed is:
 1. A process for the preparation of a compound of formula: ##STR3## wherein R represents that the substituents on the adjacent carbon atom are in the R configuration; S represents that the substituents on the adjacent carbon atom are in the S configuration; R₁ is hydrogen or hydroxy and R₂ is hydroxy or β-glucopyranosyloxy and, if R₁ is hydroxy, the C-22 atom has the S-configuration, or of a solvate or salt thereof which comprises culturing the strain So ce 12 (NCIB) 12134) of the species Sorangium cellulosum or a mutant derived from said strain that produces compounds of formula I, in a culture medium containing a source of carbon and nitrogen and essential inorganic salts, in the temperature range from about 20° to 40C. and at a pH in the range from about 4.0 to 8.0, under aerobic conditions and isolating the resultant compound into a salt and/or a resultant salt into the free compound or into another salt.
 2. A biologically pure culture of the strain So ce 12 (NCIB 12134) of the species Sorangium cellulosum or a mutant derived from said strain which produces a compound of formula in a recoverable amount ##STR4## wherein R represents that the substituents on the adjacent carbon atom are in the R configuration; S represents that the substituents on the adjacent carbon atom are in the S configuration; R₁ is hydrogen or hydroxy and R₂ is hydroxy orβ-glucopyranosyloxy and, if R₁ is hydroxy, the C-22 atom has the S-configuration or of a solvate or salt thereof. 